Compositions comprising phosphodiesterase inhabitors for the treatment of sexual disfunction

ABSTRACT

The present invention relates to highly selective phosphodieterase (PDE) enzyme inhibitors and to their use in pharmaceutical articles of manufacture. In particular, the present invention relates to potent inhibitors of cyclic guanosine 3′,5′-monophosphate specific phosphodiesterase type 5 (PDE5) that when incorporated into a pharmaceutical product at about 1 to about 20 mg unit dosage are useful for the treatment of sexual dysfunction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the U.S. national phase application of International ApplicationNo. PCT/US00/11129, filed on Apr. 26, 2000, which claims the benefit ofprovisional patent application Ser. No. 60/132,036, filed Apr. 30, 1999.

FIELD OF THE INVENTION

The present invention relates to a highly selective phosphodiesterase(PDE) enzyme inhibitor and to its use in a pharmaceutical unit dosageform. In particular, the present invention relates to a potent inhibitorof cyclic guanosine 3′,5′-monophosphate specific phosphodiesterase type5 (PDE5) that when incorporated into a pharmaceutical product is usefulfor the treatment of sexual dysfunction. The unit dosage form describedherein is characterized by selective PDE5 inhibition, and accordingly,provides a benefit in therapeutic areas where inhibition of PDE5 isdesired, with minimization or elimination of adverse side effectsresulting from inhibition of other phosphodiesterase enzymes.

BACKGROUND OF THE INVENTION

The biochemical, physiological, and clinical effects of cyclic guanosine3′,5′-monophosphate specific phosphodiesterase (cGMP-specific PDE)inhibitors suggest their utility in a variety of disease states in whichmodulation of smooth muscle, renal, hemostatic, inflammatory, and/orendocrine function is desired. Type 5 cGMP-specific phosphodiesterase(PDE5) is the major CGMP hydrolyzing lyzing enzyme in vascular smoothmuscle, and its expression in penile corpus cavernosum has been reported(Taher et al., J. Urol., 149, p. 285A (1993)). Thus, PDES is anattractive target in the treatment of sexual dysfunction (Murray, DN&P6(3), pp. 150-56 (1993)).

A pharmaceutical product, which provides a PDE5 inhibitor, is currentlyavailable and marketed under the trademark VIAGRA®. The activeingredient in VIAGRA® is sildenafil. The product is sold as an articleof manufacture including 25, 50, and 100 mg tablets of sildenafil and apackage insert. The package insert provides that sildenafil is a morepotent inhibitor of PDE5 than other known phosphodiesterases (greaterthan 80 fold for PDEl inhibition, greater than 1,000 fold for PDE2,PDE3, and PDE4 inhibition). The IC₅₀ for sildenafil against PDE5 hasbeen reported as 3 rM (Drugs of the Future, 22(2), pp. 138-143 (1997))and as 3.9 nM (Boolel et al., Int. J. of Impotence, 8, pp. 47-52(1996)). Sildenafil is described as having a 4,000-fold selectivity forPDE5 versus PDE3, and only a 10-fold selectivity for PDE5 versus PDE6.Its relative lack of selectivity for PDE6 is theorized to be the basisfor abnormalities related to color vision.

While sildenafil has obtained significant commercial success, it hasfallen short due to its significant adverse side effects, includingfacial flushing (10% incidence rate). Adverse side effects limit the useof sildenafil in patients suffering from vison abnormalities,hypertension, and, most significantly, by individuals who use organicnitrates (Welds et al., Amer. J. of Cardiology, 83(5A), pp. 21(C)-28(C)(1999)).

The use of sildenafil in patients taking organic nitrates causes aclinically significant drop in blood pressure which could place thepatient in danger. Accordingly, the package label for sildenafilprovides strict contraindications against its use in combination withorganic nitrates (e.g., nitroglycerin, isosorbide mononitrate,isosorbide nitrate, erythrityl tetranitrate) and other nitric oxidedonors in any form, either regularly or intermittently, becausesildenafil potentiates the hypotensive effects of nitrates. See C. R.Conti et al., Amer. J. of Cardiology, 83(5A), pp. 29C-34C (1999). Thus,even with the availability of sildenafil, there remains a need toidentify improved pharmaceutical products that are useful in treatingsexual dysfunction.

Daugan U.S. Pat. No. 5,859,006 discloses certain tetracyclic derivativesthat are potent inhibitors of cGMP-specific PDE, or PDES. The IC₅₀ ofthe compounds disclosed in U.S. Pat. No. 5,859,006 is reported in therange of 1 nM to 10 μM. The oral dosage for such compounds is 0.58 mgdaily for an average adult patient (70 kg). Thus, unit dosage forms(tablets or capsules) are reported as 0.2 to 400 mg of active compound.Significant adverse side effects attributed to compounds disclosed inU.S. Pat. No. 5,859,006 are not disclosed.

Applicants have discovered that one such tetracyclic derivative,(6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′,1′:6,1]pyrido[3,4-b]indole-1,4-dione,alternatively named(6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methylpyrazino-[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione,and referred to herein as Compound (I), can be administered in a unitdose that provides an effective treatment without the side effectsassociated with the presently marketed PDE5 inhibitor, sildenafil. Priorto the present invention such side effects were considered inherent tothe inhibition of PDE5.

Significantly, applicants' clinical studies also reveal that aneffective product having a reduced tendency to cause flushing insusceptible individuals can be provided. Most unexpectedly, the inproduct also can be administered with clinically insignificant sideeffects associated with the combined effects of a PDE5 inhibitor and anorganic nitrate. Thus, the contraindication once believed necessary fora product containing a PDE5 inhibitor is unnecessary when Compound (I)is administered as a unit dose of about 1 to about 20 mg, as disclosedherein. Thus, the present invention provides an effective therapyfor-sexual dysfunction in individuals who previously were untreatable orsuffered from unacceptable side effects, including individuals havingcardiovascular disease, such as in individuals requiring nitratetherapy, having suffered a myocardial infarction more than three monthsbefore the onset of sexual dysfunction therapy, and suffering from class1 congestive heart failure, or individuals suffering from visionabnormalties.

The present invention provides Compound (I) in a unit dosage form. Thatis, the present invention provides a pharmaceutical unit dosage formsuitable for oral administration comprising about 1 to about 20 mgCompound (I).

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical dosage form for humanpharmaceutical use, comprising about 1 to about 20 mg of(6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)pyrazino[2′,1′:6,1]pyrido[3,4-b]indole-1,4-dionein a unit dosage form suitable for oral administration.

The present invention further provides a method of treating conditionswhere inhibition of PDE5 is desired, which comprises administering to apatient in need thereof an oral dosage form containing about 1 to about20 mg of a selective PDE5 inhibitor, as needed, up to a total dose of 20mg per day. The invention further provides the use of an oral dosageform comprising a selective PDE5 inhibitor at a dosage of about 1 toabout 20 mg for the treatment of sexual dysfunction.

Specific conditions that can be treated by the present invention,include, but are not limited to, male erectile dysfunction and femalesexual dysfunction, particularly female arousal disorder, also known asfemale sexual arousal disorder.

In particular, the present invention is directed to a pharmaceuticalunit dosage composition comprising about 1 to about 20 mg of a compoundhaving the structural formula:

said unit dosage form suitable for oral administration, and method oftreating sexual dysfunction using the pharmaceutical unit dosecomposition.

DETAILED DESCRIPTION

For purposes of the present invention as disclosed and described herein,the following terms and abbreviations are defined as follows.

The term “container” means any receptacle and closure therefor suitablefor storing, shipping, dispensing, and/or handling a pharmaceuticalproduct.

The term “IC₅₀” is the measure of potency of a compound to inhibit aparticular PDE enzyme (e.g., PDE1c, PDE5, or PDE6). The IC₅₀ is theconcentration of a compound that results in 50% enzyme inhibition in asingle dose-response experiment. Determining the IC₅₀ value for acompound is readily carried out by a known in vitro methodologygenerally described in Y. Cheng et al., Biochem. Pharmacol., 22, pp.3099-3108 (1973).

The term “package insert” means information accompanying the productthat provides a description of how to administer the product, along withthe safety and efficacy data required to allow the physician,pharmacist, and patient to make an informed decision regarding use ofthe product. The package insert generally is regarded as the “label” fora pharmaceutical product.

The term “oral dosage form” is used in a general sense to referencepharmaceutical products administered orally. Oral dosage forms arerecognized by those skilled in the art to include such forms as liquidformulations, tablets, capsules, and gelcaps.

The term “vision abnormalities” means abnormal vision characterized byblue-green vision believed to be caused by PDE6 inhibition.

The term “flushing” means an episodic redness of the face and neckattributed to vasodilation caused by ingestion of a drug, usuallyaccompanied by a feeling of warmth over the face and neck and sometimesaccompanied by perspiration.

The term “free drug” means solid particles of drug not intimatelyembedded in a polymeric coprecipitate.

The presently claimed dosage form preferably is packaged as an articleof manufacture for human pharmaceutical use, comprising a packageinsert, a container, and a dosage form comprising about 1 to about 20 mgof Compound (I)

The package insert provides a description of how to administer apharmaceutical product, along with the safety and efficacy data requiredto allow the physician, pharmacist, and patient to make an informeddecision regarding the use of the product. The package insert generallyis regarded as the label of the pharmaceutical product. The packageinsert incorporated into the article of manufacture indicates thatCompound (I) is useful in the treatment of conditions wherein inhibitionof PDE5 is desired. The package insert also provides instructions toadminister one or more about 1 to about 20 mg unit dosage forms asneeded, up to a maximum total dose of 20 mg per day. Preferably, thedose administered is about 5 to about 20 mg/day, more preferably about 5to about 15 mg/day. Most preferably, a 10 mg dosage form is administeredonce per day.

Preferred conditions to be treated include sexual dysfunction (includingmale erectile dysfunction; and female sexual dysfunction, and morepreferably female arousal disorder (FAD)). The preferred condition to betreated is male erectile dysfunction.

Significantly, the package insert supports the use of the product totreat sexual dysfunction in patients suffering from a retinal disease,for example, diabetic retinopathy or retinitis pigmentosa, or inpatients who are using organic nitrates. Thus, the package insertpreferably is free of contraindications associated with theseconditions, and particularly the administration of the dosage form withan organic nitrate. More preferably, the package insert also is free ofany cautions or warnings both associated with retinal diseases,particularly retinitis pigmentosa, and associated with individuals proneto vision abnormalties. Preferably, the package insert also reportsincidences of flushing below 2%, preferably below 1%, and mostpreferably below 0.5%, of the patients administered the dosage form. Theincidence rate of flushing demonstrates marked improvement over priorpharmaceutical products containing a PDE5 inhibitor.

The container used in the article of manufacture is conventional in thepharmaceutical Iarts. Generally, the container is a blister pack, foilpacket, glass or plastic bottle and accompanying cap or closure, orother such article suitable for use by the patient or pharmacist.Preferably, the container is sized to accommodate 1-1000 solid dosageforms, preferably 1 to 500 solid dosage forms, and most preferably, 5 to30 solid dosage forms.

Oral dosage forms are recognized by those skilled in the art to include,for example, such forms as liquid formulations, tablets, capsules, andgelcaps. Preferably the dosage forms are solid dosage forms,particularly, tablets comprising about 1 to about 20 mg of Compound (I).Any pharmaceutically acceptable excipients for oral use are suitable forpreparation of such dosage forms. Suitable pharmaceutical dosage formsinclude coprecipitate forms described, for example, in Butler U.S. Pat.No. 5,985,326, incorporated herein by reference. In preferredembodiments, the unit dosage form of the present invention is a solidfree of a coprecipitate form of Compound (I), but rather contains solidCompound (I) as a free drug.

Preferably, the tablets comprise pharmaceutical excipients generallyrecognized as safe such as lactose, microcrystalline cellulose, starch,calcium carbonate, magnesium stearate, stearic acid, talc, and colloidalsilicon dioxide, and are prepared by standard pharmaceuticalmanufacturing techniques as described in Remington's PharmaceuticalSciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). Suchtechniques include, for example, wet granulation followed by drying,milling, and compression into tablets with or without film coating; drygranulation followed by milling, compression into tablets with orwithout film coating; dry blending followed by compression into tablets,with or without film coating; molded tablets; wet granulation, dried andfilled into gelatin capsules; dry blend filled into gelatin capsules; orsuspension and solution filled into gelatin capsules. Generally, thesolid dosage forms have identifying marks which-are debossed orimprinted on the surface.

The present invention is based on detailed experiments and clinicaltrials, and the unexpected observations that side effects previouslybelieved to be indicative of PDE5 inhibition can be reduced toclinically insignificant levels by the selection of a compound and unitdose. This unexpected observation enabled the development of a unitdosage form that incorporates Compound (I) in about 1 to about 20 mg perunit dosage forms that, when orally administered, minimizes undesirableside effects previously believed unavoidable. These side effects includefacial flushing, vision abnormalities, and a significant decrease inblood pressure, when Compound (I) is administered alone or incombination with an organic nitrate. The minimal effect of Compound (I),administered in about 1 to about 20 mg unit dosage forms, on PDE6 alsoallows the administration of a selective PDE5 inhibitor to patientssuffering from a retinal disease, like diabetic retinopathy or retinitispigmentosa.

Compound (I) has the following structural formula:

The compound of structural formula (I) was demonstrated in humanclinical studies to exert a minimal impact on systolic blood pressurewhen administered in conjunction with organic nitrates. By contrast,sildenafil demonstrates a four-fold greater decrease in systolic bloodpressure over a placebo, which leads to the contraindications in theVIAGRA insert, and in warnings to certain patients.

The following illustrates the PDE5 and PDE6 IC₅₀ values for the compoundof structural formula (I) determined by the procedures described herein.

Compound PDE5 IC₅₀ (nM) PDE6 IC₅₀ (nM) PDE6/PDE5 I 2.5 3400 1360The compound of structural formula (I) additionally demonstrates an IC₅₀against PDE1c of 10,000, and a ratio of PDE1c/PDE5 of 4,000.

PREPARATIONS

Human PDE5 Preparation

Recombinant production of human PDE5 was carried out essentially asdescribed in Example 7 of U.S. Pat. No. 5,702,936, incorporated hereinby reference, except that the yeast transformation vector employed,which is derived from the basic ADH2 plasmid described in V. Price etal., Methods in Enzymology, 1985, pages 308-318 (1990), incorporatedyeast ADH2 promoter and terminator sequences rather than ADH1 promoterand terminator sequences and the Saccharomyces cerevisiase host was theprotease-deficient strain BJ2-54 deposited on Aug. 31, 1998 with theAmerican Type Culture Collection, Manassas, Va., under accession numberATCC 74465. Transformed host cells were grown in 2×SC-leu medium, pH6.2, with trace metals, and vitamins. After 24 hours, YEP mediumcontaining glycerol was added to a final concentration of 2×YEP/3%glycerol. Approximately 24 hours later, cells were harvested, washed,and stored at −700C.

Cell pellets (29 g) were thawed on ice with an equal volume of lysisbuffer (25 mM Tris-Cl, pH 8, 5 mM MgCl₂, 0.25 mM dithiothreitol, 1 mMbenzamidine, and 10 μM ZnSO₄). Cells were lysed in a microfluidizer withN₂ at 20,000 psi. The lysate was centrifuged and filtered through 0.45μm disposable filters. The filtrate was applied to a 150 mL column of QSepharose Fast Flow (Pharmacia). The column was washed with 1.5 volumesof Buffer A (20 mM Bis-Tris Propane, pH 6.8, 1 mM MgCl₂, 0.25 mMdithiothreitol, 10 μM ZnSO₄) and eluted with a step gradient of 125 AMNaCl in Buffer A followed by a linear gradient of 125-1000 mM NaCl inBuffer A.

Active fractions from the linear gradient were applied to a 180 mLceramic hydroxyapatite column in Buffer B (20 mM Bis-Tris Propane (pH6.8), 1 MM MgCl₂, 0.25 mM dithiothreitol, 10 μM ZnSO₄, and 250 mM KCl).After loading, the column was washed with 2 volumes of Buffer B andeluted with a linear gradient of 0-125 mM potassium phosphate in BufferB. Active fractions were pooled, precipitated with 60% ammonium-sulfate,and resuspended in Buffer C (20 mM Bis-Tris Propane, pH 6.8, 125 mMNaCl, 0.5 mM dithiothreitol, and 10 μM ZnSO,). The pool was applied to a140 mL column of Sephacryl S-300 HR and eluted with Buffer C. Activefractions were diluted to 50% glycerol and stored at −20° C. Theresultant preparations were about 85% pure by SDS-PAGE.

Assay for PDE Activity

Activity of PDE5 can be measured by standard assays in the art. Forexample, specific activity of any PDE can be determined as follows. PDEassays utilizing a charcoal separation technique were performedessentially as described in Loughney et al., (1996), The Journal ofBiological Chemistry, 271:796-806. In this assay, PDE5 activity converts[³²p]cGMP to [32p]5′GMP in proportion to the amount of PDE5 activitypresent. The [³²P]5′GMP then is quantitatively converted to free [³²P]phosphate and unlabeled adenosine by the action of snake venom5′-nucleotidase. Hence, the amount of [³²P] phosphate liberated isproportional to enzyme activity. The assay is performed at 30 C in a 100μL reaction mixture containing (final concentrations) 40 mM Tris-Cl (pH8.0), 1 μM ZnSO₄, 5 mM MgCl, and 0.1 mg/mL bovine serium albumin. PDE5is present in quantities that yield <30% total hydrolysis of substrate(linear assay conditions). The assay is initiated by addition ofsubstrate (1 nM [³²P]cGMP), and the mixture is incubated for 12 minutes.Seventy-five (75) μg of Crotalus atrox venom then is added, and theincubation is continued for 3 more minutes (15 minutes total). Thereaction is stopped by addition of 200 mL of activated charcoal (25mg/mL suspension in 0.1 M NaH₂PO₄, pH 4). After centrifugation (750 ×gfor 3 minutes) to sediment the charcoal, a sample of the supernatant istaken for radioactivity determination in a scintillation counter and thePDE5 activity is calculated. The preparations had specific activities ofabout 3 μmoles cGMP hydrolyzed per minute per milligram protein.

Bovine PDE6 Preparation

Bovine PDE6 was supplied by Dr. N. Virmaux, INSERM U338, Strasbourg.Bovine retinas were prepared as described by Virmaux et al., FEBSLetters, 12(6), pp. 325-328 (1971) and see also, A. Sitaramayya et al.,Exp. Eye Res., 25, pp. 163-169 (1977). Briefly, unless stated otherwise,all operations were done in the cold and in dim red light. Eyes werekept in the cold and in the dark for up to four hours afterslaughtering.

Preparation of bovine retinal outer segment (ROS) basically followedprocedures described by Schichi et al., J. Biol. Chem., 224:529 (1969).In a typical experiment, 35 bovine retinas were ground in a mortar with35 mL 0.066 M phosphate buffer, pH 7.0, made up to 40% with sucrose,followed by homogenization in a Potter homogenizer (20 up and downstrokes). The suspension was centrifuged at 25,000×g for 20 minutes. Thepellet was homogenized in 7.5 mL 0.006 M phosphate buffer (40% insucrose), and carefully layered under 7.5 mL of phosphate buffer(containing no sucrose). Centrifugation was conducted in a swing-outrotor at 45,000×g for 20 minutes, and produced a pellet which is blackat the bottom, and also a red band at the interface 0.066 M.phosphate—40% sucrose/0.066 M phosphate (crude ROS). The red material atthe interface was removed, diluted with phosphate buffer, spun down to apellet, and redistributed in buffered 40% sucrose as described above.This procedure was repeated 2 or 3 times until no pellet was formed. Thepurified ROS was washed in phosphate buffer and finally spun down to apellet at 25,000×g for 20 minutes. All materials were then kept frozenuntil used.

Hypotonic extracts were prepared by suspending isolated ROS in 10 mMTris-Cl pH 7.5, 1 mM EDTA, and 1 mM dithioerythritol, followed bycentrifugation at 100,000×g for 30 minutes.

The preparation was reported to have a specific activity,of about 35nmoles cGMP hydrolyzed per minute per milligram protein.

PDE1c Preparation from Spodoptera fugiperda Cells (Sf9)

Cell pellets (5g) were thawed on ice with 20 ml of Lysis Buffer (50 mMMOPS pH 7.4, 10 μM ZnSO₄, 0.1 mM CaCl₂, 1 mM DTT, 2mM benzamidine HCl, 5μg/ml each of pepstatin, leupeptin, and aprotenin). Cells were lysed bypassage through a French pressure cell (SLM-Aminco) while temperatureswere maintained below 10° C. The resultant cell homogenate wascentrifuged at 36,000 rpm at 4° C. for 45 minutes in a Beckmanultracentrifuge using a Type TI45 rotor. The supernatant was discardedand the resultant pellet was resuspended with 40 ml of SolubilizationBuffer (Lysis Buffer containing 1M NaCl, 0.1M MgCl₂, 1 mM CaCl₂, 20μg/ml calmodulin, and 1% Sulfobetaine SB12 (Z3-12) by sonicating using aVibraCell tuner with a microtip for 3×30 seconds. This was performed ina crushed ice/salt mix for cooling. Following sonication, the mixturewas slowly mixed for 30 minutes at 4° C. to finish solubilizing membranebound proteins. This mixture was centrifuged in a Beckmanultracentrifuge using a type TI45 rotor at 36,000 rpm for 45 minutes.The supernatant was diluted with Lysis Buffer containing 10 μg/mlcalpain inhibitor I and II. The precipitated protein was centrifuged for20 minutes at 9,000 rpm in a Beckman JA-10 rotor. The recoveredsupernatant then was subjected to Mimetic Blue AP AgaroseChromatography.

In order to run the Mimetic Blue AP Agarose Column, the resin initiallywas shielded by the application of 10 bed volumes of 1%polyvinylpurrolidine (i.e., MW of 40,000) to block nonspecific bindingsites. The loosely bound PVP-40 was removed by washing with 10 bedvolumes of 2M NaCl, and 10 mM sodium citrate pH 3.4. Just prior toaddition of the solubilized PDE1c3 sample, the column was equilibratedwith 5 bed volumes of Column Buffer A (50 mM MOPS pH 7.4, 10 μM ZnSO₄,5mM MgCl₂, 0.1 mM CaCl₂, 1 mM DTT, 2 mM benzamidine HCl).

The solubilized sample was applied to the column at a flow rate of 2ml/min with recycling such that the total sample was applied 4 to 5times in 12 hours. After loading was completed, the column was washedwith 10 column volumes of Column Buffer A, followed by 5 column volumesof Column Buffer B (Column Buffer A containing 20 mM 5′-AMP), andfollowed by 5 column volumes of Column Buffer C (50 mM MOPS pH 7.4, 10μM ZnSO₄, 0.1 mM CaCl₂, 1 mM dithiothreitol, and 2 mM benzamidine HCl).The enzyme was eluted into three successive pools. The first poolconsisted of enzyme from a 5 bed volume wash with Column Buffer Ccontaining 1 mM cAMP. The second pool consisted of enzyme from a 10 bedvolume wash with Column Buffer C containing 1 M NaCl. The final pool ofenzyme consisted of a 5 bed volume wash with Column Buffer C containing1 M NaCl and 20 mM cAMP.

The active pools of enzyme were collected and the cyclic nucleotideremoved via conventional gel filtration chromatography or chromatographyon hydroxy-apatite resins. Following removal of cyclic nucleotides, theenzyme pools were dialyzed against Dialysis Buffer containing 25 mM MOPSpH 7.4, 10 μM ZnSO₄, 500 mM NaCl, 1 mM CaCl₂, 1 mM dithiothreitol, 1 mMbenzamidine HCl, followed by dialysis against Dialysis buffer containing50% glycerol. The enzyme was quick frozen with the aid of dry ice andstored at −70° C.

The resultant preparations were about >90% pure by SDS-PAGE. Thesepreparations had specific activities of about 0.1 to 1.0 μmol cAMPhydrolyzed per minute per milligram protein.

IC₅₀ Determinations

The parameter of interest in evaluating the potency of a competitiveenzyme inhibitor of PDE5 and/or PDE1c and PDE6 is the inhibitionconstant, i.e., K_(i). This parameter can be approximated by determiningthe ICS,, which is the inhibitor concentration that results in 50%enzyme inhibition, in a single dose-response experiment under thefollowing conditions.

The concentration of inhibitor is always much greater than theconcentration of enzyme, so that free inhibitor concentration (which isunknown) is approximated by total inhibitor concentration (which isknown).

A suitable range of inhibitor concentrations is chosen (i.e., inhibitorconcentrations at least several fold greater and several fold less thanthe K_(i) are present in the experiment). Typically, inhibitorconcentrations ranged from 10 nM to 10 μM.

The concentrations of enzyme and substrate are chosen such that lessthan 20% of the substrate is consumed in the absence of inhibitor(providing, e.g., maximum substrate hydrolysis of from 10 to 15%), sothat enzyme activity is approximately constant throughout the assay.

The concentration of substrate is less than one-tenth the Michaelisconstant (K_(m)). Under these conditions, the IC₅₀ will closelyapproximate the K_(i). This is because of the Cheng-Prusoff equationrelating these two parameters: IC₅₀=K_(i)(1+S/K_(m)), with (1+S/K_(m))approximately 1 at low values of S/K_(m).

The IC₅₀ value is estimated from the data points by fitting the datato.a suitable model of the enzyme inhibitor interaction. When thisinteraction is known to involve simple competition of the inhibitor withthe substrate, a two-parameter model can be used:Y=A/(1+x/B)where the y is the enzyme activity measured at an inhibitorconcentration of x, A is the activity in the absence of inhibitor and Bis the IC₅₀. See Y. Cheng et al., Biochem. Pharmacol., 22:3099-3108(1973).

Effects of inhibitors of the present invention on enzymatic activity ofPDE5 and PDE6 preparations as described above were assessed in either oftwo assays which differed from each other principally on the basis ofscale and provided essentially the same results in terms of IC₅₀ values.Both assays involved modification of the procedure of Wells et al.,Biochim. Biophys. Acta, 384:430 (1975). The first of the assays wasperformed in a total volume of 200 μl containing 50 mM Tris pH 7.5, 3 mMMg acetate, 1 mM EDTA, 50 μg/mL snake venom nucleotidase and 50 nM[³H]-cGMP (Amersham). Compounds of the invention were dissolved in DMSOfinally present at 2% in the assay. The assays were incubated for 30minutes at 30° C. and stopped by addition of 800 μl of 10 mM Tris pH7.5, 10 mM EDTA, 10 mM theophylline, 0.1 mM adenosine, and 0.1 mMguanosine. The mixtures were loaded on to 0.5 mL QAE Sephadex columns,and eluted with 2 mL of 0.1 M formate (pH 7.4). The eluted radioactivitywas measured by scintillation counting in Optiphase Hisafe 3.

A second, microplate, PDE assay was developed using Multiscreen platesand a vacuum manifold. The assay (100 μl ) contained 50 mM Tris pH 7.5,5 mM Mg acetate, 1 mM EDTA and 250 μg/mL snake venom nucleotidase. Theother components of the reaction mixture were as described above. At theend of the incubation, the total volume of the assays were loaded on aQAE Sephadex microcolumn plate by filtration. Free radioactivity waseluted with 200 μl of water from which 50 μl aliquots were analyzed byscintillatio n counting as described above.

The following examples are presented to further illustrate thepreparation of the claimed invention. The scope of the present inventionis not to be construed as merely consisting of the following examples.

EXAMPLE 1

Compound (I) was prepared as described in U.S. Pat. No. 5,859,006 andformulated in tablets using wet granulation. Povidone was dissolved inwater to make a 10% solution. The active compound, microcrystallinecellulose, croscarmellose sodium, and sodium lauryl sulfate were addedto a high shear mixer and mixed for 2 minutes. The powders were wetgranulated with the povidone solution and extra water as required tocomplete the granulation. The resultant mixture was dried in a fluid beddrier with inlet air at 70° C.±5° C. until the loss on drying was below2.5%. The granules were passed through a Comil with a suitable screen(or a sieve) and added to a suitable mixer. The extragranularcroscarmellose sodium and sodium lauryl sulfate, and the colloidalanhydrous silica were passed through a suitable sieve (e.g., 500 micron)and added to the mixer and blended 5 minutes. Magnesium stearate wasadded and blended for 2 minutes. The blend was compressed to a targetcompression/weight of 250 mg using 9 mm round normal concave tooling.

The core tablets were coated with an aqueous suspension of OpadryOY-S-7322 using an Accelacota (or similar coating pan) using inlet airat 50° C. to 70° C. until the tablet weight was increased byapproximately 8 mg. Opadry OY-S-7322 containsmethylhydroxypropylcellulose Ph. Eur., titanium dioxide Ph. Eur.,Triacetin USP. Opadry increases the weight of each tablet to about 258mg. The amount of film coat applied per tablet may be less than thatstated depending on the process efficiency.

The tablets are filled into blister packs and accompanied by packageinsert describing the safety and efficacy of the compound.

Formulations Component (mg per tablet) Selective PDE5 Inhibitor¹⁾ 1 5Hydroxypropyl Methylcellulose 1 5 Phthalate Microcrystalline Cellulose221.87 213.87 Croscarmellose Sodium 5.00 5.00 Sodium Lauryl Sulfate 2.502.50 Povidone K30 9.38 9.38 Purified Water, USP (water for q.s. q.s.irrigation) Croscarmellose Sodium 5.00 5.00 Sodium Lauryl Sulfate 2.502.50 Colloidal Anhydrous Silica 0.50 0.50 Magnesium Stearate 1.25 1.25Total core subtotal 250.00 250.00 (Film coat Opadry OY-S-7322) about 8mg about 8 mg ¹⁾Compound (I).

EXAMPLE 2

The following formula is used in preparing the finished dosage formcontaining 10 mg of Compound (I).

Ingredient Quantity (mg) Granulation Selective PDE5 Inhibitor¹⁾ 10.00Lactose Monohydrate 153.80 Lactose Monohydrate (spray dried) 25.00Hydroxypropylcellulose 4.00 Croscarmellose Sodium 9.00Hydroxypropylcellulose (EF) 1.75 Sodium Lauryl Sulfate 0.70 35.00Outside Powders Microcrystalline Cellulose (granular-102) 37.50Croscarmellose Sodium 7.00 Magnesium Stearate (vegetable) 1.25 Total 250mg Film coat (approximately) 11.25

Purified Water, USP is used in the manufacture of the tablets. The wateris removed during processing and minimal levels remain in the finishedproduct.

Tablets are manufactured using a wet granulation process. A step-by-stepdescription of the process is as follows. The drug and excipients to begranulated are security sieved. The selective PDE5 inhibitor is dryblended with lactose monohydrate (spray dried), hydroxypropylcellulose,croscarmellulose sodium, and lactose monohydrate. The resulting powderblend is granulated with an aqueous solution of hydroxypropylcelluloseand sodium lauryl sulfate using a Powrex or other suitable high sheargranulator. Additional water can be added to reach the desired endpoint.A mill can be used to delump the wet granulation and facilitate drying.The wet granulation is dried using either a fluid bed dryer or a dryingoven. Once the material is dried, it can be sized to eliminate any largeagglomerates. Microcrystalline cellulose, croscarmellose sodium, andmagnesium stearate are security sieved and added to the dry sizedgranules. These excipients and the dry granulation are mixed untiluniform using a tumble bin, ribbon mixer, or other suitable mixingequipment. The mixing process can be separated into two phases. Themicrocrystalline cellulose, croscarmellose sodium, and the driedgranulation are added to the mixer and blended during the first phase,followed by the addition of the magnesium stearate to this granulationand a second mixing phase.

The mixed granulation then is compressed into tablets using a rotarycompression machine. The core tablets are film coated with an aqueoussuspension of the appropriate color mixture in a coating pan (e.g.,Accela Cota). The coated tablets can be lightly dusted with talc toimprove tablet handling characteristics.

The tablets are filled into plastic containers (30 tablets/container)and accompanied by package insert describing the safety and efficacy ofthe compound.

EXAMPLE 3

The following formula is used in preparing a finished dosage formcontaining 5 mg of Compound (I).

Ingredient Quantity (mg) Granulation Selective PDE5 Inhibitor¹⁾ 2.50Lactose Monohydrate 79.395 Lactose Monohydrate (spray dried) 12.50Hydroxypropylcellulose 2.00 Croscarmellose Sodium 4.50Hydroxypropylcellulose (EF) 0.875 Sodium Lauryl Sulfate 0.35 OutsidePowders Microcrystalline Cellulose (granular-102) 18.75 CroscarmelloseSodium 3.50 Magnesium Stearate (vegetable) 0.63 Total 125 mg Film coat(approximately) 6.875

The dosage form of Example 3 was prepared in an identical manner to thedosage form of Example 2.

EXAMPLE 4

Solution Capsule Ingredient mg/capsule Percent (%) Selective PDE5Inhibitor¹⁾ 10 2 PEG400 NF 490 98 Fill Weight 500 100

The gelatin capsules are precisely filled by pumping an accurate fillvolume of pre-dissolved drug formulation into the partially sealedcavity of a capsule. Immediately following injection fill of the drugsolution formulation, the capsule is completely heat sealed.

The capsules are filled into plastic containers and accompanied by apackage insert.

EXAMPLE 5

This study was a randomized, double-blind, placebo-controlled, two-waycrossover design clinical pharmacology drug interaction study thatevaluated the hemodynamic effects of concomitant administration of aselective PDES inhibitor (i.e., Compound (I)) and short-acting nitrateson healthy male volunteers. In this study, the subjects received eitherCompound (I) at a dose of 10 mg or a placebo, daily for seven days. Onthe sixth or seventh day, the subjects received sublingual nitroglycerin(0.4 mg) while supine on a tilt table. The nitroglycerin wasadministered 3 hours after Compound (I) dosing, and all subjects keptthe nitroglycerine tablet under their tongue until it completelydissolved. The subjects were tilted to 70° head-up every 5 minutes for atotal of 30 minutes with measurement of blood pressure and heart rate.There were no discontinuations among the twenty-two healthy malesubjects (ages 19 to 60 years old) that entered this study.

In a preliminary analysis of this study, Compound (I) was well toleratedand there were no serious adverse events. There were no Compound (I)changes in laboratory safety assessments or 12-lead ECGs. The mostcommon adverse events were headache, dyspepsia, and back pain. Compound(I) demonstrated minimal, if any, effect on mean systolic bloodpressure, and mean maximal nitroglycerin-induced decrease in systolicblood pressure.

EXAMPLE 6

In two randomized, double-blinded placebo controlled studies, Compound(I) was administered to patients in need thereof at a range of doses, inboth daily dosing and for on demand therapy, for sexual encounters andintercourse in the home setting. Doses from 5 to 20 mg of Compound (I)were efficacious and demonstrated less than 1% flushing and no reportsof vision abnormalities. It was found that a 10 mg dose of Compound (I)was fully efficacious and demonstrated minimal side effects.

Enhanced erectile function was determined by the International Index ofErectile Function (IIEF) (Rosen et al., Urology, 49, pp. 822-830(1997)), diaries of sexual attempts, and a global satisfaction question.Compound (I) significantly improved the percentage of successfulintercourse attempts including the ability to attain and maintain anerection in both “on demand” and daily dosing regimens.

EXAMPLE 7

A third clinical study was a randomized, double-blind,placebo-controlled study of Compound (I) administered “on demand” topatients with male erectile dysfunction. Compound (I) was administeredover a period of eight weeks in the treatment of male erectiledysfunction (ED). Erectile dysfunction (ED) is defined as the persistentinability to attain and/or maintain an erection adequate to permitsatisfactory sexual performance. “On demand” dosing is defined asintermittent administration of Compound (I) prior to expected sexualactivity.

The study population consisted of 212 men, at least 18 years of age,with mild to severe erectile dysfunction. Compound (I) was orallyadministered as tablets of coprecipitate made in accordance with ButlerU.S. Pat. No. 5,985,326. Compound (I) was administered in 2 mg, 5 mg, 10mg, and 25 mg doses, “on demand” and not more than once every 24 hours.Treatment with all nitrates, azole antifungals (e.g., ketoconazole oritraconazole), warfarin, erythromycin, or antiandrogens was not allowedat any time during the study. No other approved or experimentalmedications, treatments, or devices used to treat ED were allowed.Forty-one subjects were administered a placebo.

The two primary efficacy variables were the ability of a subject topenetrate his partner and his ability to maintain an erection duringintercourse, as measured by the International Index of Erectile Function(IIEF). The IIEF Questionnaire contains fifteen questions, and is abrief, reliable measure of erectile function. See R. C. Rosen et al.,Urology, 49, pp. 822-830 (1997)

Secondary efficacy variables were IIEF domain scores for erectilefunction, orgasmic function, sexual desire, intercourse satisfaction,and overall satisfaction; the patient's ability to achieve an erection,ability to insert his penis into his partner's vagina, completion ofintercourse with ejaculation, satisfaction with the hardness of hiserection, and overall satisfaction, all as measured by the SexualEncounter Profile (SEP) diary; and a global assessment question asked atthe end of the treatment period. The SEP is a patient diary instrumentdocumenting each sexual encounter during the course of the study.

The safety aspect of the study included all enrolled subjects, and wasassessed by evaluating all reported adverse events, and changes inclinical laboratory values, vital signs, physical examination results,and electrocardiogram results.

At endpoint, patients who rated their penetration ability (IIEF Question3) as “almost always or always” were as follows: 17.5% in the placebogroup, 38.1% in the 2 mg group, 48.8% in the 5 mg group, 51.2% in the 10mg group, and 83.7% in the 25 mg group. Comparisons revealedstatistically significant differences in change in penetration abilitybetween placebo and all dose levels of Compound (I).

At endpoint, patients who rated their ability to maintain an erection(IIEF Question 4) during intercourse as “almost always or always” are asfollows: 10.0% in the placebo group, 19.5% in the 2 mg group, 32.6% inthe 5 mg group, 39.0% in the 10 mg group, and 69.0% in the 25 mg group.Comparison revealed statistically significant differences in change inpenetration ability between placebo and the three higher dose levels ofCompound (I).

This study also included a safety evaluation. A treatment-emergentadverse event is defined as a condition not present at baseline thatappeared postbaseline, or a condition present at baseline that increasedin severity postbaseline. The most commonly reported treatment-emergentadverse events were headache, dyspepsia, and back pain. The incidence oftreatment-emergent adverse events appeared related to dose.

Overall, this study demonstrated that all four doses of Compound (I),namely 2 mg, 5 mg, 10 mg, and 25 mg, taken “on demand” producedsignificant improvement, relative to placebo, in the sexual performanceof men with erectile dysfunction as assessed by the IIEF, by patientdiaries assessing frequency of successful intercourse and intercoursesatisfaction, and by a global assessment.

The combined results from clinical studies showed that administration ofCompound (I) effectively treats male erectile dysfunction, asillustrated in the following table.

IIEF ERECTILE FUNCTION DOMAIN (Change from Baseline) Unit Dose ofCompound (I) n Mean ± SD p placebo 131 0.8 ± 5.3  2 mg 75 3.9 ± 6.1<.001  5 mg 79 6.6 ± 7.1 <.001 10 mg 135 7.9 ± 6.7 <.001 25 mg 132 9.4 ±7.0 <.001 50 mg 52 9.8 ± 5.5 <.001 100 mg  49 8.4 ± 6.1 <.001 n isnumber of subjects, SD is standard deviation.

However, it also was observed from the combined clinical studies thatthe percent of treatment-emergent adverse events increased with anincreasing unit dose of Compound (I), as illustrated in the followingtable:

Treatment-Emergent Adverse Events (%) Unit Dose of Compound (I) (mg)Event Placebo 2 5 10 25 50 100 Headache 10 12 10 23 29 34 46 Dyspepsia 63 14 13 19 20 25 Back Pain 5 3 3 15 18 24 22 Myalgia 3 0 3 9 16 20 29Rhinitis 3 7 3 4 4 0 2 Conjunctivitis 1 0 1 1 0 2 5 Eyelid Edema 0 0 0 11 2 3 Flushing 0 0 0 <1 0 3 7 Vision 0 0 0 0 0 0 0 AbnormalitiesThe above table shows an increase in adverse events at 25 mg through 100mg unit doses. Accordingly, even though efficacy in the treatment of EDwas observed at 25 mg to 100 mg doses, the adverse events observed from25 mg to 100 mg doses must be considered.

In accordance with the present invention, a unit dose of about 1 toabout 20 mg, preferably about 2 to about 20 mg, more preferably about 5to about 20 mg, and most preferably about 5 to about 15 mg, of Compound(I), administered up to a maximum of 20 mg per 24-hour period, botheffectively treats ED and minimizes or eliminates the occurrence ofadverse side effects. importantly, no vision abnormalities were reportedand flushing was essentially eliminated. Surprisingly, in addition totreating ED, with at about 1 to about 20 mg unit dose Compound (I), witha minimum of adverse side effects, individuals undergoing nitratetherapy also can be treated for ED by the method and composition of thepresent invention.

The principles, preferred embodiments, and modes of operation of thepresent invention have been described in the foregoing specification.The invention intended to be protected herein, however, is not construedto be limited to the particular forms disclosed, because they are to beregarded as illustrative rather than restrictive. Variations and changesmay be made by those skilled in the art without departing from thespirit of the invention.

1. A method of treating sexual dysfunction in a patient in need thereofcomprising orally administering one or more unit dose containing about 1to about 20 mg, up to a maximum total dose of 20 mg per day, of acompound having the structure


2. The method of claim 1 wherein the sexual dysfunction is male erectiledysfunction.
 3. The method of claim 1 wherein the sexual dysfunction isfemale arousal disorder.
 4. The method of claim 1 wherein the unit dosecontains about 2 to about 20 mg of the compound.
 5. The method of claim1 wherein the unit dose contains about 5 mg of the compound.
 6. Themethod of claim 1 wherein the unit dose contains about 10 mg of thecompound and is administered once per day.
 7. The method of claim 1wherein the unit dose is in a form selected from the group consisting ofa liquid, a tablet, a capsule, and a gelcap.
 8. The method of claim 1wherein the unit dose contains about 2.5 mg of the compound.
 9. Themethod of claim 8 wherein the unit dose is administered once per day.10. The method of claim 5 wherein the unit dose is administered once perday.
 11. The method of claim 1 wherein the compound is administered as afree drug.
 12. The method of claim 1 wherein the unit dose containsabout 20 mg of the compound.